DESIGNING A SHUTTLE VECTOR FOR PROTEIN PRODUCTION IN PICHIA PASTORIS - - PowerPoint PPT Presentation

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Mar 21, 2023 439 likes •603 views

DESIGNING A SHUTTLE VECTOR FOR PROTEIN PRODUCTION IN PICHIA PASTORIS pastoris i PICHIA Georgia State University School Background Georgia State University was founded as an extension of the Georgia School of Technologys Evening

SLIDE 1

DESIGNING A SHUTTLE VECTOR FOR PROTEIN PRODUCTION IN PICHIA PASTORIS

Georgia State University

iPICHIA pastoris

SLIDE 2

School Background

 Georgia State University was

founded as an extension of the Georgia School of Technology‟s “Evening School of Commerce” back in 1913

 It has now become the second

largest research institution in the University System of Georgia

 GSU „s has a 34 acre campus in

the heart of Downtown Atlanta

 This year GSU got a record high

enrollment of above 30,000 students

 This is GSU‟s second year

participating in iGEM.

Petite. H Parker Science Building

SLIDE 3

Introduction

 Historically two organisms have been most commonly used as

hosts for recombinant protein production

 Escherichia coli Saccharomyces cerevisiae

 Limitations of S. cerevisiae

 Hyperglycosylation  Low yields

 Limitations of E. coli

 No post – translational

modifications

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Advantages of Using Pichia pastoris

 Inexpensive to culture  High production of foreign proteins  Post-translational modification capacity  Strongly inducible promoters

AOXI and AOXII

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Promoter

 Alcohol Oxidase I promoter

Strongly inducible promoter

 Activated by methanol  Inactivated in the presence of glucose

Controls expression of Alcohol Oxidase

Methanol Formaldehyde + Hydrogen Peroxide

SLIDE 6

Primer Design

 Forward primer

5‟ 3‟

 Reverse primer

5‟ 3‟

 Isolated Gene

EcoRI NotI XbaI PstI NotI SpeI EcoI NotI XbaI SpeI NotI PstI

SLIDE 7

Isolation of Alcohol Oxidase I

 Alcohol Oxidase I

promoter gene is 940bp

 Addition of the restriction

sites using primers should add 40bp

 As seen in the figure a

band was present slightly below 1000bp indicating that AOX1 was successfully isolated.

SLIDE 8

Vector Design

 Alcohol Oxidase I

Promoter

Rep pMB1origin

BBa_K165058

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Vector Design

Rep pMB1 origin

Zeocin Resistance BspH1 EarI

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Vector Design

Rep pMB1 origin

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Homologous Recombination

Integration of the vector into the chromosome of P . pastoris in a single cross over event

SLIDE 12

Future Applications

 Characterize the Alcohol Oxidase promoter

 Fluorescence of the yeast

 Swap with Pichia promoters

 Example

 Gly-3-Phosphate Dehydrogenase  Formaldehyde Dehydrogenase  Transcription Elongation Factor  Production of eukaryotic proteins and production of

vaccines

SLIDE 13

2011 Team Accomplishments

 Established GSU Synthetic Biology Club  Created 3-semester iGEM course for future

development

 Received $45,000 departmental funding for lab

equipment

 Hosted multiple fundraising events  Kaplan Auction  Bake Sales  Keg Parties  Racquetball Tournament

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Acknowledgements

 Dr. Sidney Crow  Dr. George Pierce  Dr. Malcolm Zellars  Dr. Eric Gilbert  Dr. Paul Ulrich  Sarah Boyd  Chris Cornelison  GSU Synthetic Biology

Club

 KAPLAN  Anatolia Café